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Full text of "Screening maize for resistance to giberella ear rot."

■*■ 



Agriculture and Agriculture et 

Agri-Food Canada Agroalimentaire Canada 



Research 
Branch 



Direr H 
de 



H ♦ I A 9 ricuItur9 



Canada 



c-3 



Canadian Agriculture Library 
Bibliotheque canadienne de I'agriculture 
Ottawa K1 A 0C5 



%JUL 1 7 
JUIL I I 



Screening Maize 

for Resistance to 

Gibberella Ear Rot 




c .3 



Screening Maize 

for Resistance to 

Gibberella Ear Rot 



L.M. Reid and R.I. Hamilton 
Eastern Cereal and Oilseed Research Centre 

Ottawa, Ontario 

D.E. Mather 

Macdonald Campus, McGill University 

Ste-Anne-de-Bellevue, Quebec 

Technical Bulletin 1996- 5E 



Research Branch 
Agriculture and Agri-Food Canada 

1996 



& 



Eastern Cereal and Oilseed Centre de recherches de l'Est 
Research Centre sur les cereales et oleagineux 



Copies of this publication are available from 

Director 

Eastern Cereal and Oilseed Research Centre 

Research Branch, Agriculture and Agri-Food Canada 

K.W. Neatby Building 

Ottawa, Ont.Kl A 0C6 

Produced by Eastern Cereal and Oilseed Research Centre 

© Minister of Supply and Services Canada 1996 
Cat. No.A54-8/1996-5E 
ISBN 0-662-24595-4 
Printed 1996 



Contents 



Acknowledgments 


5 


Preface 


5 


Summary 


7 


Resume 


7 


Introduction 


9 


Silk vs. Kernel Inoculations 


13 


Inoculum Production 


15 


Number of Isolates 


16 


Silk Channel Inoculation 


17 


Inoculation Site 


17 


Inoculum Volume 


19 


Inoculum Concentration 


20 


Timing of Inoculation 


20 


Summary of Instructions for Silk Channel Inoculations 


22 


Kernel Inoculation 


24 


Inoculation Site 


26 


Inoculum Concentration 


26 


Timing of Inoculation 


27 


Summary of Instructions for Kernel Inoculations 


28 


Disease Severity Assessments 


30 


Visual Rating Scales 


30 


Selection of Resistant Plants 


32 



Mycotoxin Content 32 

Plot Maintenance 33 

Experimental Design and Statistical Analyses 34 

Safety - Handling of Conidial Suspensions and Infected Ears 35 
References 37 



Acknowledgments 

We acknowledge the significant contribution of Alf Bolton 
who laid the foundation of this work. We are indebted to 
N. Brown, T. Woldemariam, and Y. Chen for technical support. 
We thank A. Schaafsma of the Ontario Ministry of Agriculture 
and Food and Rural Affairs for suggesting the use of the 
backpacks. The reviews and editorial suggestions of D. Born, 
C. Chungu, L. Harris, C. Ide, F. Meloche, J.D. Miller, T. Ouellet, 
R. Pandeya, L. Seaman, B. Vigier, and T. Woldemariam are 
appreciated. The authors thank Pioneer Hi-Bred, the Natural 
Sciences and Engineering Research Council of Canada, the 
Ontario Pork Producers Association, and the Ontario Corn 
Producers Association for financial support at various points 
throughout our research. We greatly appreciate all of the hard 
work and effort Judy McCarthy and her co-workers Alice 
Whelan, Eric Johnson and Lise St-Jean put into the graphical 
presentation of this bulletin. 



Preface 

Since 1986, scientists of the maize (corn) improvement 
program of the Eastern Cereal and Oilseed Research Centre 
(ECORC) (formally the Plant Research Centre), Agriculture and 
Agri-Food Canada, have been developing field-plot techniques 
to screen maize inbreds and hybrids for resistance to ear mold 
caused by Fusarium graminearum. Two techniques were 
developed, one to screen for resistance to infection via the silk 
and one to screen for resistance to infection via kernel wounds. 
Both techniques have undergone testing to standardize them for 
routine use in breeding programs and pathology research. 
These techniques allow for good differentiation between 
genotypes, ranging from very susceptible to highly resistant and 
are now being used in maize breeding programs to develop 
inbred lines with improved resistance to F. graminearum. In 
addition, both techniques have been used successfully to infect 
maize ears with F. moniliforme, F. subglutinans, and F. culmorum. 

Although this booklet is designed primarily to assist 
researchers in maize breeding, the techniques described have 
been used to study resistance mechanisms, inheritance of 
resistance, epidemiology and basic pathology of this important 
maize disease. 



Digitized by the Internet Archive 
in 2013 



http://archive.org/details/screeningmaizefo19965reid 



Summary 

The development of suitable techniques for the screening and 
evaluation of maize for resistance to gibberella ear rot could 
greatly facilitate breeding for resistant hybrids. Development of 
such techniques requires many studies and rigorous testing. This 
review describes two field screening techniques which have been 
standardized for routine use in breeding programs. The 
techniques are differentiated on the basis of the mode of fungal 
entry simulated: silk vs. kernel infection. The silk channel 
inoculation technique consists of injecting a Fusarium graminearum 
macroconidial suspension into the silk channel of primary ears. 
The kernel inoculation technique consists of stabbing the centre of 
the ear with four stainless steel pins previously dipped in a 
macroconidial suspension. Details of inoculum production, 
inoculation site, inoculum concentration and volume, timing of 
inoculation, plot maintenance, statistical analyses, evaluation and 
interpretation of results, mycotoxin analysis, and safety 
procedures are discussed. 

Resume 

La selection genetique d'hybrides de mais resistants peut etre 
amelioree par le developpement de techniques adaptees pour 
1'evaluation et l'identification de lignees de mais resistantes a la 
fusariose (gibberella) de l'epi. La conception de telles techniques 
necessite de nombreuses etudes et une evaluation rigoureuse des 
resultats. Ce bulletin decrit deux techniques de demarquage au 
champs qui doivent faire partie integrante d'un programme de 
selection pour Amelioration du mais resistant a la fusariose de 
l'epi. Ces techniques se distinguent sur la base de deux modes de 
simulation d'attaques fongiques, soit une infection via les soies de 
l'epi ou bien une infection directe sur les grains. La technique 
d'inoculation par le col des soies consiste en une injection d'une 
suspension de macroconidies de Fusarium graminearum appliquee 
sur l'epi principal. Le type d'inoculation par les grains consiste en 
un marquage, au moyen d'un pioncpn muni de quatre pointes en 
acier inoxidable, au centre de l'epe et au travers de grains, qui a 
ete trempe dans une solution macroconidienne. Les differentes 
etapes de ces techniques sont discutees, a savoir, la production 
d'inoculant, le choix du site d'essai, le volume et la concentration 
de l'inoculant, la cedule d'inoculation, l'entretien des parcelles, les 
analyses statistiques et l'interpretation des resultats, les analyses 
de mycotoxines et enfin les normes de securite a respecter. 



Introduction 

Fusarium graminearum Schwabe, the asexual state of Gibberella 
zeae (Schw.) Petch, is an important ear-rotting pathogen of maize 
(corn) in many areas of the world, including Canada (Gordon 
1959, Sutton et al. 1980a), the United States (Hesseltine and 
Bothast 1977, Koehler 1959), southern and eastern Europe (Milic 
et al. 1969), central and southern Africa (Marasas et al. 1979), the 
former USSR (Manannikova 1979), and China (Tanaka et al. 1988). 
The disease itself is called gibberella ear rot or pink mold. 
Fusarium graminearum is also a causal agent of stalk rot in maize 
and head blight (scab) in wheat. 

The major symptom of F. graminearum infection on maize ears 
is a characteristic pink- to reddish-coloured mold on kernels and 
between husks and kernels (Fig. 1). Silks and husks may adhere 
tightly to the kernels in severely infected ears, and mold growth 
may be visible on husks at the tip of the ear. Kernel infection is 
usually found near the tip of the ear or around tunnels made by 
insect feeding. If the season is long and wet, small, round, black 
perithecia (fruiting structure in which ascospores are formed) of 
G. zeae may develop on the surface of infected husks. 

Growth of F. graminearum requires periods of warm 
temperatures (optimum 24°C-26°C) with persistent wetness. 
Rainfall and warm temperatures during July and August (silking 
and early kernel development) are key factors in epidemics of 
gibberella ear rot (Koehler 1959, Miller 1994, Sutton 1982, Tuite et 
al. 1974). The major sources of F. graminearum inoculum seem to 
be infested host debris such as old stalks and ears of maize or 
debris from a preceding wheat crop (Sutton 1982). Such refuse 
may give rise directly to infectious mycelium, or may serve as a 
food base for sporulation and dissemination. Nearby infected 
wheat fields can also be sources of inoculum. The use of infected 
grain as seed results in poor stands and diseased seedlings 
(blight). During epidemics, it is believed that the major means of 
inoculum dispersal is aerial; ascospores and macroconidia are 
therefore the most important inoculum types. Birds and insects 
can also be vectors of F. graminearum and wounds created by 
feeding may predispose the ear to further fungal invasion 
(Attwater and Busch 1983, Enerson and Hunter 1980, Sutton et al. 
1980b). Damage of ears by hail has also been found to increase 
the incidence of infection (Abbas et al. 1988). Fungal entry into 
maize ears can occur through two major modes: (1) by growth of 
mycelium down silks to the kernels and cob (rachis) from spores 




Fig. 1. Maize ear infected with Fusarium graminearum, 
exhibiting the discolouration of husk (A), mycelial growth 
between husk leaves (B), and characteristic pink to reddish- 
coloured mold on kernels (C) associated with gibberella ear rot. 



10 



germinating on the silks; and (2) by entry through wounds 
(Hesseltine and Bothast 1977, Koehler 1942, Sutton 1982). 

Although the incidence and severity of gibberella ear rot can 
be somewhat sporadic and localized from year to year, ear rot 
does reduce the total yielding potential of hybrids and losses in 
grain quality may be appreciable due to mycotoxins produced by 
this pathogen. This is of considerable concern to livestock 
producers. Swine are the most sensitive to F. graminearum 
mycotoxins. Two major mycotoxins are produced by this 
pathogen: zearalenone and deoxynivalenol. Zearalenone causes 
swine estrogenic syndrome, as well as male infertility, reduced 
litter size, feed refusal, and haemorrhagia (Mirocha and 
Christensen 1974, Prelusky et al. 1994). The trichothecene toxin 
deoxynivalenol (DON, vomitoxin) causes vomiting, feed refusal, 
and decreased weight gain in swine (Prelusky et al. 1994, 
Vesonder et al. 1981). Trichothecenes are also 
immunosuppressants and inhibitors of protein synthesis and 
thus can predispose animals to other diseases and mask 
underlying toxicoses (Pestka and Bondy 1990). Besides causing 
direct and indirect economic losses, this fungus can also affect 
the health of grain handlers and processors. It is therefore 
imperative to develop and devise adequate control and 
protection measures against this disease. 

Various management strategies which give some degree of ear 
rot control include plowing under of residues, weed control, crop 
rotation with non-graminaceous crops, and balanced soil fertility. 
Once infection has taken place, various strategies might be tried 
to reduce further fungal growth and contamination; these 
include proper grain drying, storage of grain at low moisture 
levels, and sanitation of feed preparation and delivery systems 
(Enerson and Hunter 1980, Martin and Johnston 1982, Shurtleff 
1984). Dilution of contaminated grain with clean grain has been 
used but it is not a fully satisfactory control method and is often 
not practical for growers who produce their own feeds 
(Charmley and Prelusky 1994). Decontamination of grain by 
chemical treatment is not economically feasible. Since the 
pathogen utilizes the maize matrix, infected kernels are 
significantly lighter than uninfected ones, and thus density 
segregation can be used to remove damaged kernels at harvest. 
Lighter infected kernels can be removed when combine- 
harvesting, thus potentially reducing the toxin content in the 
harvested grain (Charmley and Prelusky 1994, Trenholm et al. 
1988). A considerable proportion of deoxynivalenol is in the cob 



11 



or rachis and thus will be removed at harvest when cobs are 
discarded (Reid et al. 1996a). 

The best way to control gibberella ear rot is to prevent 
infection in the field. This can be achieved with the development 
of resistant hybrids through genetic improvement and breeding. 
It is the most economical and efficient means of controlling maize 
diseases and is the control measure that is most readily accepted 
by growers. 

Availability of reliable screening methods for detecting 
resistance is the cornerstone of any disease resistance breeding 
program. One might consider using mycotoxin levels in the 
grain to screen for resistance. However, chemical analysis of 
mycotoxin levels is very time-consuming and expensive. 
Mycotoxin assay kits based on monoclonal antibodies have been 
developed for qualitative assays of individual toxins and are 
faster and cheaper than chemical analyses of mycotoxins. 
However, these kits are not yet inexpensive enough to use in a 
breeding program. 

Currently, the only way to screen for resistance to gibberella 
ear rot is in the field. There are two reasons for this: (1) 
satisfactory levels of infection and reliable genotypic 
differentiation have not been achieved under greenhouse 
conditions; and (2) there is no laboratory technique or seedling 
test that can be used to screen for the type of resistance that is 
exhibited in a fully grown field plant. 

Due to the sporadic nature of gibberella ear rot epidemics, 
artificial inoculation techniques are needed to enhance 
incubation and infection and to overcome variability of infection 
during years when natural contamination is too low to identify 
genotypic differences. This variability has been a major limiting 
factor in breeding for resistance since methods of inoculation 
and screening have not been consistent. A diversity of results, 
many of which are conflicting, have been obtained with different 
inoculation and post-inoculation treatments. Growth stage of 
the host, inoculum type, position of inoculum, wounding of ear, 
and environmental conditions are among the factors affecting 
development and expression of disease. 

The development and evaluation of any inoculation and 
selection technique for gibberella ear rot resistance involves a 
number of essential steps: 



12 



1 . Develop an appropriate method of maintaining the 
pathogen in pure culture to provide inoculum. 

2. Determine the most efficient and reproducible method of 
applying the inoculum to the plant. 

3. Determine the inoculum dose or density required to produce 
the desired amount of infection. 

4. Determine the part of the ear to be inoculated. 

5. Determine the optimum stage of plant growth or time for 
inoculation since disease incidence and severity can be 
affected by the maturity^of the host plant. 

6. Determine if different isolates of the pathogen differ in 
aggressivity, and whether there are important genotype by 
isolate interactions. 

7. Control the field environment whenever possible to produce 
uniform and optimum infection and disease development, 
e.g. by providing overhead irrigation during the incubation 
period. 

8. Maintain appropriate management practices to ensure that 
only healthy plants are used for testing. 

9. Determine the optimum number of plants and replicates 
required to give statistically significant results. 

10. Develop an accurate scale or index for measuring the 
response of plants to infection. 

The most important consideration is the effectiveness of the 
technique in demonstrating differences in disease reactions 
among genotypes for the type of resistance (silk vs. kernel) 
being assessed. 

Silk vs. Kernel Inoculations 

Evaluations of maize genotypes for resistance to gibberella 
ear rot should take into account the two modes of fungal entry, 
i.e. growth down the silks and entry through kernel wounds. 
Resistance to one mode of infection does not imply resistance to 
the other mode. Silk resistance alone is not sufficient since 
infections through the kernel can occur at most stages of ear 
development. Kernel resistance alone may not be sufficient 
since infections through the silk when kernels are not yet fully 
developed can result in extensive infection of kernels and cobs, 
(Reid and Hamilton 1996a). 



13 



Screening for silk resistance has usually involved one of three 
techniques: (1) insertion of a colonized substrate (e.g. toothpicks 
or cereal kernels overgrown with mycelium) or a pipecleaner 
impregnated with macroconidia into the silk channel (region 
within the husk between the tip of the cob and tip of the husk 
where the silks emerge); (2) spraying a conidial suspension on 
the exposed silks; or (3) injection of a conidial suspension into 
the silk channel. 

The use of colonized substrates does not simulate natural 
infection since the inoculum source is mycelium, not spores. 
Colonized substrates or pipecleaners placed in the silk channel 
are often displaced away from the ear tip as silks elongate, 
resulting in a low level of infection (Sutton and Baliko 1981). 
Spraying of a conidial suspension on silks more closely 
simulates natural infection. This technique augments natural 
infection by providing for a greater volume, a higher 
concentration of spores, a more specific time of application, and 
a more uniform distribution of inoculum. Unfortunately, low 
levels of infection are achieved with this technique. To 
counteract this, spraying of a conidial suspension on silks is 
often followed by bagging of the ear to prevent desiccation. 
However, bagging often results in excess water on ear surfaces, 
encouraging bacterial growth and reducing the level of 
infection. Injection of a conidial suspension into the silk channel 
has been found to give the most consistent results of the three 
techniques. 

Screening for kernel or wound resistance often involves 
puncturing the husk, kernels, and cob followed by insertion of a 
colonized substrate (toothpick) or spores (saturated pipecleaner) 
into the wound. High levels of infection are obtained when a 
colonized toothpick is inserted into the centre of the ear and it 
thus is difficult to distinguish resistant from susceptible 
genotypes. The insertion of toothpicks or spore-impregnated 
pipecleaners through the husk, kernels and cob, circumvents 
any physical barriers or resistance factors that could otherwise 
exclude the pathogen. Techniques which produce a point source 
wound to kernels and not to the cob more closely approximate 
infection from insect damage. Recently, methods have been 
developed to avoid wounding the cob by puncturing just the 
husk and kernels followed by application of a conidial 
suspension (Chungu et al. 1996, Reid and Hamilton 1996a, 
Schaafsma et al. 1993). 



14 



The Eastern Cereal and Oilseed Research Centre has 
developed two screening techniques, one to evaluate resistance 
to infection through the silk and one for infection through 
wounded kernels. Both techniques allow for good 
differentiation between genotypes and have been successfully 
used to identify genotypes with extremely high levels of 
resistance to either silk or kernel infection (Reid et al. 1995b). 
The following sections describe in detail the application of these 
techniques and the interpretation of the results. 



Inoculum Production 

Macroconidia are the easiest inoculum propagules of 
F. graminearum to produce in mass quantities. The technique 
and medium used for producing macroconidia depends on the 
facilities available and the amount of inoculum required. 
Ultimately, a liquid suspension of conidia is required. Best 
fungal growth is achieved in a low-sugar liquid medium that is 
subjected to some agitation (shaking, rotating, bubbling) to 
prevent mycelial growth and clumping. 

To produce a macroconidial suspension of F. graminearum, 
liquid medium consisting of the following can be used: 

2.0 g potassium dihydrogen phosphate (KH 2 P0 4 ) 

2.0 g potassium nitrate (KN0 3 ) 

1.0 g potassium chloride (KC1) 

1.0 g magnesium sulphate (MgS0 4 ) 

0.0002 g/L each of: ferric sulphate (FeS0 4 ), ferric chloride 
(FeCl 3 ), manganese sulphate (MnS0 4 ), and zinc 
sulphate (ZnS0 4 ) 

1 .0 L distilled water 

1 .0 g dextrose 

Other carbohydrate sources, such as 2.0 g of soluble starch or 
sucrose, can also be used. The medium is dispensed at 150 mL 
into 500 mL erlenmeyer flasks, autoclaved for 20 minutes 
(pH=5), then a 1 cm 2 piece of potato dextrose (PDA) agar 
containing mycelium and macroconidia of a single isolate of 
F. graminearum is added. Cultures are shaken at 25°C for 1 hr at 
4 hr intervals under natural light supplemented with fluorescent 
light [Sylvania Cool White (F40 CW); GTE. Corp.]. Conidial 
concentrations can reach 2 x 10 6 spores /mL in one week 



15 



depending on strain. Prepared inoculum can be stored at 2-4°C 
(refrigerator) for a maximum of four weeks before decreases in 
spore viability occur. Prior to inoculation, the mixture is filtered 
through two layers of cheesecloth to remove mycelial clumps 
and diluted with sterile water to the desired conidial 
concentration. A typical flask of 150 mL of 2 x 10 6 conidia/mL 
suspension when diluted to a concentration of 5 x 10 5 
conidia/mL (final volume of 450 mL) will provide enough 
inoculum to inoculate 225 plants at 2 mL per plant. 

Sterile water is used for a control since by the time of 
inoculation the inoculum largely consists of spore suspension in 
diluted spent medium. Sedimentation or centrifugation of the 
conidia and resuspension in sterile water, to eliminate the 
addition of any medium to the inoculated ear, is not 
recommended since most of the conidia will lyse in such 
conditions and little infection is achieved. 

Number of Isolates 

Some F. graminearum isolates may be more aggressive than 
others, inducing greater disease severity. There may also be some 
interaction between maize genotypes and pathogen isolates, but 
most isolates will consistently differentiate or rank the most 
resistant and the most susceptible genotypes. Inconsistencies in 
ranking tend to be restricted to those genotypes with moderate 
susceptibility (Atlin et al. 1983, Mesterhazy and Kovacs 1986, Reid 
et al. 1993). If a single sufficiently aggressive isolate is used, a 
plant breeder will be able to identify genotypes with useful 
resistance and those with severe susceptibility. If a mixture of 
isolates is used, conidial suspensions of each isolate are prepared 
and mixed just before inoculation. 

Any isolate(s) used for screening should have been isolated 
originally from naturally infected maize ears and should be able to 
cause typical ear rot symptoms in susceptible maize and produce 
mycotoxins in infected kernels. Virulent F. graminearum isolates 
can be obtained from the Canadian Collection of Fungus Cultures, 
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri- 
Food Canada, Ottawa, Ontario, Kl A OC6. Isolates should be 
freeze-dried or frozen in liquid nitrogen for storage. 



16 



Silk Channel Inoculation 

The most satisfactory silk inoculation technique involves the 
injection of a conidial suspension of F. graminearum into the silk 
channel (Fig. 2) inside the husk cavity and above the cob. In a 
breeding nursery, pollinations are conducted as usual, then ear 
shoot bags can be lifted to perform inoculations and replaced. 

Two mL of inoculum are injected into the silk channel of each 
primary ear using a graduated, 10 mL, self-refilling, automatic 
vaccinator attached to a 2.5 L backpack container (Nasco Co., 
Fort Atkinson, WI) (Fig. 3). An 18-gauge Luer-lock stainless steel 
hypodermic needle is attached to the vaccinator. One individual 
can inoculate 300-400 ears per hour. Secondary ears are not 
inoculated since they are not present in all genotypes and they 
often mature later than primary ears. 



Fig. 2 . Longitudinal 
section of a maize ear 
showing location of silk 
channel. 




Stalk 
internode - 

Ear node 



Silk 

Silk 
channel 

Kernels 

Husk leaves 
Shank 



Inoculation Site 

For kernels to become infected with the silk channel 
technique, macroconidia placed within the silk channel must 
germinate and hyphae must grow down the silks to infect 
developing kernels. The rate of progression of the fungus down 
the silk channel is a function of the degree of inherent silk 
resistance, silk age, and environment. For example, if silk 
resistance is not complete but is sufficient to delay the 
progression of the fungus down the silk until the kernels have 
hardened then infection will be minimal. Once the fungus 



17 



reaches the kernels, the severity of infection is a function of the 
inherent resistance of the kernels, kernel maturity, and 
environment. This inoculation technique cannot be used for 
genotypes with little or no silk channel. 

To successfully assess silk resistance, care should be taken to 
ensure that the inoculator needle is held at right angles to the 
silk channel (Fig. 3C), otherwise macroconidia will be injected 




Fig. 3. Silk channel inoculation of maize ears with a 
macroconidial suspension of Fusarium graminearum using a 
graduated, self-refilling, automatic vaccinator (A) attached to a 
2.5 L backpack (B). Two mL of suspension is injected into the 
silk channel while holding the vaccinator at right angles to the 
long axis of the ear (C). 



18 



down the silk channel onto the kernels. If this happens disease 
severity ratings will be high and a measurement of resistance to 
infection via the silk will not be obtained. Injections are made 
into the centre of the silk channel, which can be estimated by 
feeling for the tip of the cob and injecting halfway between that 
point and the end of the silk channel, where the silks emerge 
from the husk. Care should be taken to avoid wounding too 
low, as this would flood the kernels and possibly wound the cob 
tip. Occasionally the needle may become plugged with silk and 
husk tissue. This can be avoided by periodically forcibly 
squeezing the vaccinator trigger and injecting inoculum into a 
waste container to dislodge any tissue. 

Inoculum Volume 

The silk channel cavity of most maize genotypes will hold 
about 2 mL of inoculum. Higher volumes of inoculum 
significantly increase the amount of infection in more susceptible 
genotypes (Fig. 4). This is largely due to the probability that 
with higher volumes, inoculum will be forced down the silk 
channel reaching the cob tip and kernels, thus overcoming the 
natural barrier of the silk and any silk resistance. Genotypes 
with useful silk resistance will therefore, not be selected as they 
will appear susceptible. Moreover, the ability to differentiate 
between genotypes will be reduced and selection will be 
ineffective. 



Fig. 4. Effect of 
inoculum volume on 
gibberella ear rot 
disease severity for two 
maize hybrids 
inoculated with the silk 
channel technique. 
Disease severity ratings 

arp nn a 1—7 sralp whprp 


7 
6 - 

> 
(D 
CO 
CD 4 - 

CO 
CD 

Q 3 - 

2 - 
1 


_. - — ~* Susceptible 

/ 

/ 

/ 


1= no infection and 7= 
>75% of the kernels 


^^^'^ Resistant 


visibly moldy 
(see Fig. 11). 




i i i i i i i i i i 

1 23456789 10 

Conidial suspension volume (ml) 



19 



Inoculum Concentration 

A concentration of 5 x 10 5 spores /mL has been observed to 
give maximum differentiation among genotypes, ranging from 
resistant to very susceptible, although most concentrations will 
differentiate between the most resistant and the most susceptible 
(Reid et al. 1995a). Higher concentrations significantly increase 
the amount of infection in susceptible genotypes (Fig. 5), 
especially in years with overall higher levels of infection. Lower 
concentrations will also differentiate between genotypes, but the 
degree of differentiation may be lower in years which are less 
conducive to fungal growth. 



5 3 

2 

1 



Susceptible , 



esistant ^^^ 

m — *r 



103 104 105 106 

Conidial suspension concentration (log) (conidia/ml) 



Fig. 5. Effect of 
inoculum concentration 
on gibberella ear rot 
disease severity for two 
maize hybrids 
inoculated with the silk 
channel technique (2 mL 
volume). Disease 
severity ratings are on a 
1-7 scale where 1= no 
infection and 7= >75% 
of the kernels visibly 
moldy (see Fig. 11). 



Timing of Inoculation 

Stage of plant growth at inoculation is the most important 
technical parameter to consider for gibberella ear rot (Reid et al. 
1992). Silk channel inoculations are best if done 4-7 days after 
silk emergence when there is a peak in expression of 
susceptibility (Fig. 6). This period corresponds to the stage 
when silks are elongated, pollinated and may have some tip 
browning but are still green (not dry). The silks of maize 
senesce rapidly after pollination and this physiological change 
seems to alter the suitability of silk for growth of ear-rotting 
organisms. Infections are insufficient when inoculations are 
made more than 8 days after silk emergence, especially if the 
silks have dried out; assessments are then incorrect or no 
differentiation is observed. Timing is critically important when 



20 



Fig. 6. Effect of time of 
inoculation after silk 
emergence on gibberella 
ear rot disease severity 
for two maize hybrids 
inoculated with the silk 
channel technique. 
Disease severity ratings 
are on a 1-7 scale where 
1= no infection and 7= 
>75% of the kernels 
visibly moldy 
(see Fig. 11). 



■E 5 

(D 

> 
CD 

w 4 

<D 

CO 

CO 

CD 

S£ 3 

Q 



Susceptible 




2 4 6 8 10 12 

Inoculation time (days after silking) 



16 



18 



genotypes with different maturity are to be evaluated. Timing 
can be based on the number of days from 50% silk emergence 
(50% of the plants of a given genotype with emerged silk) or, in 
large screening programs where this is not feasible, timing can 
be based on the physical appearance of the silk. 

Timing of inoculation among plants of a given genotype is 
also important. More consistent results will be obtained if all 
ears of a given genotype are inoculated at the same time, so that 
the environmental conditions at time of inoculation are 
consistent. Rows 3.8 m long with 14 plants are convenient and 
all plants in the row are inoculated when 10 of the centre 12 
plants are ready for inoculation. If the rows are from a 
segregating population, this is not possible and the row should 
be visited several times until all desired plants are inoculated. 
To keep track of which plants have been inoculated and to avoid 
double-inoculations or escapes, a small dot of spray paint (red is 
easiest to see at harvest) is sprayed onto the lower husk/ shank 
area to mark the inoculated plants. 



21 



Summary of Instructions for Silk Channel Inoculations 

The following steps describe the inoculation of maize silk 
channels with a macroconidial suspension of F graminearum 
using a self-refilling automated vaccinator attached to a 2 L 
backpack: 

1 . Prepare conidial suspension at least one week in advance 
of date of inoculation. 

2. Assemble vaccinator by attaching rubber hose and clamps. 
Assemble backpack by attaching straps. Thoroughly rinse 
vaccinator, hose and backpack with 70% ethanol, then with 
sterile water. Let dry. 

Do a spore count of conidial suspension and dilute to 
5 x 10 5 spores/mL with sterile water. Use rubber gloves for 
this step and the rest of the procedure (AVOID ALL 
CONTACT WITH SPORE SUSPENSION). 

4. Filter through 2 layers of cheesecloth. 

5. Fill backpack to volume of inoculum required (number of 
plants to be inoculated x 2 mL). 

6. Attach vaccinator and rubber hose to backpack. Ensure that 
all connections are tight to prevent leakage. 

7. Invert backpack to allow suspension to flow down the hose 
to the vaccinator Remove all air bubbles from hose and 
vaccinator by pumping suspension through the vaccinator. 
Sterilize Luer-lock needle in 70% ethanol and attach to 
vaccinator. 

8. Ensure that vaccinator is set at 2 mL by injecting into a 
graduated cylinder to check volume. Ensure that screw on 
top of vaccinator is tight to prevent volume changes (this 
screw may loosen on older vaccinators, it should be 
periodically checked while in use). 

9. Select plants to be inoculated (THIS IS THE MOST CRITICAL 
STEP). These plants will have primary ears with silk 
approximately one week old, i.e. silk is elongated and has 
some tip browning (DO NOT INOCULATE PLANTS IN 
WHICH THE SILK IS BROWN AND DRY). If experiments 
consists of genotypes of many different maturities, walk 
through the field every 3 days and record which rows have 
50% of the plants with silk emerged, these rows are then 
inoculated approximately 5-6 days later (note: this time 
span will be reduced if the growing season is rapid, so do 
not rely on chronological time alone, combine it with 
observations on silk appearance). When inoculating hybrids 



22 



or inbreds, inoculate ail of the plants that are ready in a 
row on the same day (do not go back and inoculate slower 
growing plants since this will create too much variability in 
the results). When inoculating segregating populations 
where plants are of different maturities, the row(s) should 
be visited every 2-3 days and only plants that are ready 
are inoculated. In a nursery, it is more difficult to 
determine inoculation time based on silk appearance 
since ears are covered with paper bags to prevent 
pollination. In this case, mark the pollination date on the 
bag with a water-proof marker when making pollinations 
then return to those plants 5-6 days later, lift bags, 
inoculate, then replace bags. 

10. On the primary ears (usually the highest ear on the stalk) 
of each plant, locate the centre of the silk channel by 
feeling for the tip of the cob and insert the vaccinator 
needle at a right angle to the silk channel in the midpoint 
between the cob tip and the point where the silks emerge 
from the husk. If any resistance to penetration is felt, the 
needle has been inserted into the cob and wounded it; 
move to the next ear and be careful in locating the silk 
channel. This inoculation technique cannot be used for 
genotypes with little or no silk channel. 

11 . Slowly inject the suspension into the silk channel, keeping 
the needle at right angles to the channel. 

12. Spray a small dot of red paint on the lower husk area or 
shank of the ear. If inoculation of an entire row is 
completed this can be indicated by spraying an additional 
band of paint on the internode below the tassel on the first 
plant of the row. In a nursery, since the ear is covered with 
a bag, inoculated plants can be marked by either spraying 
paint on the stalk above the ear or spraying paint directly 
on the bag. Do not inoculate a row and then come back 
and spray paint on inoculated plants, this leads to errors. 
Nursery aprons are used to carry bottles, inoculators, 
spray paint, etc. 

13. Inoculate the next plant. Every 5-6 rows, check the vaccinator to 
ensure that no blockage of the needle is occurring by rapidly 
squeezing the trigger to dislodge any tissue. When the 
inoculator is not in use, e.g. at breaks or lunch, do not leave it 
outside in the sun and heat. 

14. When finished, thoroughly rinse vaccinator and backpack 
with 70% ethanol and sterile water. Allow to air dry 
overnight. 



23 



Kernel Inoculation 

The kernel inoculation technique (Reid and Hamilton 1996a) 
involves wounding the husk and kernels by stabbing them with 
four small (3 mm dia.) stainless steel pins mounted in a 
rectangular 7 mm x 5 mm pattern on one end of a 2.5 cm x 50 cm 
cylindrical wooden handle (Fig. 7A). The pin spacing is 
designed to increase the chance of wounding four kernels when 
the inoculator is held such that the 7 mm spacing is horizontal. 
Prior to wounding, the pins are dipped in a conidial suspension. 
The conidia are carried into the wounds with the pins or by 
capillary action. Alternatively, a more expensive apparatus can 
be used to inject the kernels with inoculum (Fig. 7B). Our 
results have shown no significant difference in disease severity 
between the two techniques, but the automated apparatus has 
two advantages, it is faster to use, and it minimizes exposure of 
the user to inoculum. With both inoculators, only 3-4 kernels 
are wounded, thus creating a point source of inoculation from 
which the spread of infection from wounded to non-wounded 
kernels can be measured. One individual can inoculate an 
average of 200-300 ears per hour with this technique. As with 
the silk channel technique, inoculation can be conducted 
following pollination just by lifting the ear shoot bag. 

The depth of puncture (pin length, 5 mm) is suitable for most 
commercial maize hybrids. Care should be taken to avoid 
wounding the cob; if this happens, the infection may remain in 
the cob causing a spongy rot with no visible symptoms apparent 
on the kernels. Such an ear may be falsely evaluated as 
resistant. Alternatively, if the cob is wounded the infection may 
spread to non-wounded kernels through the cob rather than 
from wounded to non-wounded kernels. If this technique is 
used on inbreds or genotypes with thin husks, it is 
recommended that inoculations first be tested on a few border 
plants to make sure that penetration depth is appropriate. To 
adjust the depth, a small piece of rubber can be used to shorten 
the length of the pins. Penetration depth is adjustable on the 
automated inoculator (Fig. 7B). As with the silk channel 
technique, only the primary ears are inoculated and spray paint 
can be used to mark treated plants. 



24 




Fig. 7. Two methods of kernel inoculation of maize ears with 
a macroconidial suspension of Fusariutn graminearum. (A) 
Inoculator has four stainless steel pins attached to a cylindrical 
wooden handle. The pins are dipped in the suspension before 
stabbing the centre of the ear through the husk and into 3-4 
kernels, thus producing a point source of infection from which 
the fungus spreads. (B) Automatic, self-refilling, graduated 
kernel inoculator which injects inoculum into 3-4 kernels. 
Each pin has two holes drilled on the sides to prevent plugging 
upon wounding. Penetration depth is adjustable. 



25 



Inoculation Site 

As with the silk channel technique, the position of the inoculator 
is very important. The pins should enter at a right angle to the long 
axis of the ear midway between the butt and the tip. Wounds made 
too high, near the ear tip, may result more often in wounding of 
the cob than in infection of the kernels, which are less well 
developed than those at the centre of the ear (Fig. 8). Inoculations 
made too close to the butt also result in reduced infection levels, 
probably because kernel hardening progresses from the butt to the 
tip; and the more mature butt kernels are less receptive to infection. 
Inoculation in the centre of the ear produces the most uniform 
results. In susceptible genotypes, infection will rapidly spread to 
neighbouring non- wounded kernels, often forming a ring around 
the ear before progressing to tip and butt areas. Again, because the 
butt kernels harden first, infection tends to spread to the tip area 
more than the butt (Reid and Hamilton 1996a). 



CD 
> 
CD 
CO 
CD 

CO 
CD 

.2 3 



4- 



Butt 

Centre 

Tip 




I 



Resistant 



Susceptible 



Fig. 8. Effect of 
inoculation site (butt, 
centre, or tip of ear) on 
gibberella ear rot disease 
severity for two hybrids 
inoculated with the 
kernel wound technique. 
Disease severity ratings 
are on a 1-7 scale where 
1= no infection and 7= 
>75% of the kernels 
visibly moldy 
(see Fig. 11). 



Inoculum Concentration 

As with the silk channel technique, increasing the concentration 
of the conidial suspension increases disease severity (Reid and 
Hamilton 1996a) (Fig. 9). There is little difference in severity 
ratings at concentrations from 10 4 to 5 x 10 5 conidia/mL, mainly 
because the kernel technique bypasses any morphological 
resistance barriers. However, as with the silk channel technique, 
the degree of differentiation may be lower in some years which are 
less conducive to fungal growth at lower inoculum concentrations. 
For simplicity, one can use the same concentration recommended 
for the silk channel technique, i.e. 5 x 10 5 conidia/mL. 



26 



Fig. 9. Effect of 
inoculum concentration 
on gibberella ear rot 
disease severity for two 
maize hybrids 
inoculated with the 
kernel wound technique. 
Disease severity ratings 
are on a 1-7 scale where 
1= no infection and 7= 
>75% of the kernels 
visibly moldy 
(see Fig. 11). 



* 3 



Susceptible 



Resistant 



103 104 105 106 

Conidial suspension concentration (log) (conidia/mL) 



Timing of Inoculation 

Inoculating at the optimum stage of plant growth is as 
important with kernel inoculations as with the silk channel 
inoculations (Fig. 10). Kernel inoculations are made 10-15 days 
post-silk emergence. This corresponds roughly to the 'blister to 
early milk stages' of kernel development. Early inoculations 
result in too much infection since kernels are not yet developed 
and have a much lower resistance. Inoculations made too late 
result in little infection since kernels have hardened. Thus, care 
should be taken to ensure that inoculations are made at the proper 
time of kernel development and for all plants of a given genotype 
at the same time. 



Fig. 10. Effect of time 
of inoculation after silk 
emergence on gibberella 
ear rot disease severity 
for two maize hybrids 
inoculated with the 
kernel wound 
technique. Disease 
severity ratings are on a 
1-7 scale where 1= no 
infection and 7= >75% 
of the kernels visibly 
moldy (see Fig. 11). 



CD 
> 
CD 

W 4 
CD 

n3 
CD 

5 



3 - 



Susceptible 




Resistant 



8 16 24 32 40 

Inoculation time (days after silking) 



27 



Summary of Instructions for Kernel Inoculations 

The following steps describe the inoculation of maize 
kernels with a macroconidial suspension of F. graminearum 
by stabbing the ear with four small (3 mm dia.) stainless 
steel pins: 

1 . Prepare conidial suspension at least one week in 
advance of date of inoculation. 

2 Clean inoculator and a small (approx. 100 mL) plastic 
bottle with a screw cap with 70% ethanol, then with 
sterile water. Let dry. 

3. Do a spore count of conidial suspension and dilute to 

5 x 10 5 spores/mL with sterile water. Use rubber gloves 
for this step and the rest of the procedure (AVOID ALL 
CONTACT WITH SPORE SUSPENSION). 

4. Filter through 2 layers of cheesecloth. 

5 Fill plastic bottle with suspension and cap tightly. 

6. Select plants to be inoculated (THIS IS THE MOST 
CRITICAL STEP). These plants will have primary ears 
with silk approximately 10-15 days old, i.e. the silk will 
appear brown and almost dry, the ear will be thicker and 
rounder in the centre and butt due to kernel and cob 
development, and the kernels will be in the blister-early 
milk stage of development. If experiments consist of 
genotypes of many different maturities, walk through 
the field every 3 days and record which rows have 50% 
of the plants with silk emerged; these rows are then 
inoculated approximately 10-15 days later (note: this 
time span will be reduced if conditions are favourable 
for rapid plant growth so do not rely on chronological 
time alone, combine it with ear appearance). When 
inoculating hybrids or inbreds, inoculate all of the 
plants that are ready in a row on the same day (do not 
go back and inoculate slower growing plants since this 
will create too much variability in the results). When 
inoculating segregating populations where plants are of 
different maturities, the row(s) should be visited every 
2-3 days and only plants that are ready are inoculated. 
In a nursery, it is more difficult to determine inoculation 
time based on ear appearance since ears are covered 
with paper bags to prevent pollination. In this case, 



28 



mark the pollination date on the bag with a waterproof 
marker when making pollinations then return to those 
plants 5-6 days later, lift bags, inoculate, then replace 
bags. 

7. On the primary ear (usually the highest ear on the stalk) 
of each plant, locate the midpoint of the ear by feeling 
for the tip and butt of the cob and inoculating through 
the husk at that point. 

8. Unscrew the bottle with suspension and dip the 
inoculator into it. Immediately stab the inoculator into 
the midpoint of the ear at a right angle to the ear. The 
inoculator is inserted such that the 7 mm spacing 
between the pins is horizontal. Withdraw the inoculator 
smoothly. Do not wiggle the inoculator or do anything to 
increase the degree of wounding. Do not stab to the 
point where the cob is wounded, only wound the 
kernels, i.e. once the slightest bit of resistance to entry 
is felt you have hit the cob; try to avoid this by practising 
on border plants or rows. Nursery aprons are useful to 
carry bottles, inoculators, spray paint, etc. 

9. Spray a small dot of red paint on the lower husk area or 
shank of the ear, far enough away from the wound so 
that no paint enters the wound. If inoculation of an 
entire row is completed this can be indicated by 
spraying an additional band of paint on the internode 
below the tassel on the first plant of the row. In a 
nursery, since the ear is covered with a bag, inoculated 
plants can be marked by either spraying paint on the 
stalk above the ear or directly on the bag. Do not 
inoculate a row and then come back and spray paint on 
inoculated plants, this leads to errors. 

10. Inoculate the next plant. When the inoculator is not in 
use, e.g. during breaks or lunch, do not leave the 
suspension bottle outside in the sun and heat. 

11. When finished, thoroughly rinse the inoculator and 
bottle with 70% ethanol and sterile water. Allow to air 
dry overnight. 

When you are using the automated kernel inoculator 
follow the steps outlined above but fill the inoculator bottle 
with suspension and set the volume to 0.5 mL. Stab the 
plant as usual and slowly inject the suspension. 



29 



Disease Severity Assessments 

Visual Rating Scales 

Various methods have been used to evaluate the severity of 
gibberella ear rot after inoculation, including whole-row ratings 
(Koehler 1942), kernel sorting (Koehler 1959), kernel plating 
(Koehler 1959), and individual ear ratings using visual scales 
(Ullstrup 1970, Sutton and Baliko 1981, Enerson and Hunter 
1980). The latter method is the most widely used since it is the 
least time-consuming, increments on the scale are easily 
discernable, data recording is simplified, and it allows for rapid 
screening of a large number of genotypes. 

Ears are harvested at normal grain harvesting moisture 
(approximately 24%) in October. For most genotypes, it takes 6-8 
weeks for disease severity levels to reach a peak and stabilize. 
Ears are hand husked and the severity of ear rot symptoms is 
evaluated using rating scales based on the percentage of kernels 
with visible symptoms of infection such as rot and mycelial 
growth. The scales consist of the same 7 classes for both the silk 
channel (Fig. 11 A) and the kernel technique (Fig. 11B). This 
allows for direct comparisons between the two techniques. It is 
easiest and most efficient to husk the ears while they are still 
attached to the plants. After rating disease severity, ears needed 
for mycotoxin analysis can be hand-picked and the remaining 
ears removed for disposal with a combine /forage harvester. 

Providing personnel have been adequately trained and 
inoculations are properly performed, we have not found 
significant differences in severity ratings among individuals 
using the techniques described. However, more consistent 
results are achieved when one individual inoculates a given 
experiment. If experiments are large and require two or more 
individuals to inoculate, try to put each person on a separate 
replicate or block. In addition, it may be useful to conduct a 
smaller experiment in which the treatment effect is the various 
individuals. This will give a measure of the variability between 
people's techniques. However, if individuals are properly 
trained and the inoculation techniques are properly 
demonstrated, this kind of variability is minimal to not 
significant. Since interpretation of the rating scale may vary 
slightly between users, it may require periodic standardization. 
It is recommended that one person does all of the ratings within 
nurseries or experiments. 



30 



(A) Silk Channel 



(B) Kernel 



Rating 



Rating 




76-100% 



Fig. 11. Disease severity rating scales and percentage of 
visibly infected kernels for gibberella ear rot after silk 
channel (A) and kernel (B) inoculations with Fusarium 
gr amine arum. 



31 



Selection of Resistant Plants 

In a breeding program, the acceptable level of visible infection 
for selection of resistant plants is largely dependent on the 
inoculation technique used. In the case of silk channel 
inoculation, ears with no visible infection (rating=l) reflect 
resistance to the spread of infection down the silk channel. Ears 
with infected kernels imply that silk resistance was not present 
or was not sufficient to stop the fungus from reaching the kernels 
before kernel resistance developed and /or kernels hardened and 
were no longer receptive. The environment can also delay 
progression of the fungus down the silk channel, hence the need 
for further study using kernel inoculations. 

With kernel wound inoculation techniques, there is always 
some infection. In this case a resistant plant would be one in 
which the infection does not spread from the wounded kernels to 
non- wounded kernels (rating=2). This can be manifested as a 
shrinking /abortion of the wounded kernels with or without 
visible signs of infection such as mycelial growth. 



Mycotoxin Content 

A strong positive relationship (r>0.80, p<0.01) exists between 
visible disease symptoms and DON levels for both silk channel 
and kernel inoculations for most genotypes (Reid et al. 1996a,b). 
The relationship is exponential in that after a rating of 
approximately 5 (50% infection) the amount of toxin in the 
kernels can be very high. This is mostly dependent on the 
environment. Thus, mycotoxin analyses are not needed during 
routine screenings. Such a relationship has also been reported by 
other researchers using wound inoculation techniques (Atlin et 
al. 1983, Cullen et al. 1983, Hart et al. 1982, Hart et al. 1987). 
Selection can be based on visual evaluation of disease symptoms. 
This is easy and rapid and is significantly less expensive than 
mycotoxin analysis. However, mycotoxin analyses are desirable 
in the final stages of inbred and /or hybrid development before 
variety release because the acceptable level of DON in feed is low 
(in the order of 1 ppm). 

For deoxynivalenol analyses, ears are grouped on a per row 
basis (usually a bulk of 10), bagged in mesh sacks, air-dried for 
two weeks, and frozen (-20°C) until analysis. Ears (bulk) are 
hand-shelled and kernels are mixed thoroughly to obtain a 
random distribution. A 50 g sample is ground to a fine powder 



32 



in a Retsch Ultra Centrifugal Mill Type ZM1 (Brinkman 
Instruments, Rexdale, Ontario) with a 0.75 mm mesh. A sample 
of this ground tissue is then sent to the appropriate laboratories 
for mycotoxin analyses. 



Plot Maintenance 

In disease resistance studies the environment should be 
controlled as much as possible to produce uniform plant 
emergence and growth as well as optimum infection and disease 
development. This is especially important for gibberella ear rot 
because of the sporadic nature of this disease and its susceptibility 
to environmental factors. 

A humid environment should be maintained using irrigation, 
at a rate of 2-5 mm daily for the four- week period after 
inoculation. However, this may not be essential except in areas or 
years which are very dry. Irrigation may increase disease severity 
with silk channel inoculations (Fig. 12 A); but the degree of 
increase, if any, is a factor of the precipitation and temperature for 
the month after inoculation (Reid and Hamilton 1996a). In 
contrast, irrigation has little significant effect and will sometimes 
decrease infection with the kernel inoculation technique (Fig. 
12B). This may be due to penetration of the inoculum into a 
substrate since the wounds bypass morphological barriers. In 
contrast, silk channel inoculation places the fungus in a silk 
containing cavity from which the mycelium must grow along the 
silk to infect the ovules /cob, and the ability to do so may be 
influenced by the moisture content of the silk and the 
environment. A decrease in disease severity with kernel 
inoculation under irrigation may be due to water washing the 
conidia from the ear, out of the wound, before colonization takes 
place. 

It is important to conduct appropriate management practices to 
ensure that only healthy plants are inoculated. This includes 
proper fertilization, plant spacing and weed control. Stressed 
plants may have increased disease severity and resistance may 
not be apparent. Fields with uniform soil should be used to 
minimize variation in plant development since inoculations of the 
same genotype made at different times may result in variable 
results because of environmental changes. Within-plot variability 
is significantly reduced if care is taken to make plants as uniform 
as possible and if timing of inoculation is consistent. 



33 



6- 



> 5 



a? 

■c 

03 

> 
(0 
CO 
CD 

to 

03 
CD 
22 

b 



B 



H1 



Irrigated 
Non-irrigated 



_ 









H1 



H1 H1 H1 

Hybrid code 



H1 



H1 



Fig. 12. Effect of 
irrigation on gibberella 
ear rot disease severity 
for seven maize 
hybrids inoculated 
with the silk channel 
technique (A) and the 
kernel wound 
technique (B). Disease 
severity ratings are on 
a 1-7 scale where 1= no 
infection and 7= >75% 
of the kernels visibly 
moldy (see Fig. 11). 



If the inoculated ears are left on the plant following disease 
assessment, a combine can be used to remove the grain for 
disposal. Plant debris left in the field should be plowed under 
to minimize winter survival of the fungus and reduce future 
inoculum potential. 



Experimental Design and Statistical Analyses 

Ears are rated individually and a mean rating is calculated for 
each row within each of four replicates; however, methods will 
vary depending on research objectives, e.g. individual plant 
ratings may be more desirable with segregating populations. 
Entries are planted in single-row plots (3.8 m long) of 12-14 
plants. The primary ears of the centre 10 plants are inoculated. 



34 



A randomized complete block design is usually used and data are 
analyzed and presented as a range in resistance or a ranking of 
genotypes. Relatively good reproduction of infection ratings have 
been obtained from year to year. The different levels of resistance 
demonstrated in inoculated hybrids used as checks correlate well 
with those observed in the field following natural infection. 

Since a rating scale is used for assessing disease severity there 
are various methods one can choose for analysis of the data. 
Nonparametric analyses can be performed; however, they can be 
limiting in the degree of analyses available. The data can be 
transformed and then analyzed, or standard parametric statistics 
can be performed on the data provided residual error terms are 
distributed normally. 

Due to environmental influences, the ability to differentiate 
among genotypes may vary somewhat from year to year, making 
it desirable to evaluate material over two or more years. Mean 
ratings for a given genotype vary from year to year, but rankings 
among genotypes, seldom vary, even across environments/ 
locations in a given year. For example, years with average 
temperatures, following inoculation, of less than 25°C result in 
low levels of severity thus a susceptible genotype which rated 6 in 
a warm year may rate 4 in a cool year, and a resistant genotype 
may rate 2 in a warm year and 1 in a cool year. Thus it is 
important to use check genotypes that show similar rankings 
under different environments and it is important to test error 
mean squares for homogeneity before pooling data over years. 



Safety - Handling of Conidial Suspensions 
and Infected Ears 

The spores produced by F. graminearum, like those of most 
molds, can cause allergies and inflammation of lung tissue and 
these spores also contain the same mycotoxins found in the 
infected ears. These mycotoxins are toxic to humans and 
inhalation of spores or dust from contaminated grain can be 
extremely hazardous. Precautions should be taken to minimize 
contact with cultures and infected plant material by any route 
(oral, inhalation, or skin). 

Handling F. graminearum cultures, especially petri dish cultures, 
should only be done in a biological containment hood which 
draws air away from the user and through a filter. Conidial 



35 



suspensions may contain mycotoxins, so disposable rubber gloves 
and other protective clothing (labcoat, coveralls) should be worn 
when these suspensions are being filtered, diluted and placed into 
backpacks for silk channel inoculation. Once in the backpack, 
exposure to the inoculum is minimal. However, some inoculum 
may drip from the needle during injection , so gloves should be 
worn in the field. With the kernel inoculation technique, 
exposure is more probable as the inoculator is dipped into the 
conidial suspension, and again gloves are the best protection. If 
the Jet Pipet kernel inoculator is used, exposure is significantly 
reduced but gloves should still be worn. Inoculating equipment 
should be cleaned with 70% ethanol after use each day. If 
inoculations also are being conducted with other Fusarium 
species, e.g. F. moniliforme or F. subglutinans, designate an 
inoculator for each species to avoid contamination. All laboratory 
surfaces, where culture or suspension transfers have been made, 
should also be cleaned with 70% ethanol or other disinfectants 
immediately afteruse. 

The Canadian Grain Commission, Health Canada, Human 
Resources Development Canada-Labour Program, and 
Agriculture and Agri-Food Canada have established guidelines 
for the handling of grain contaminated by F. graminearum. The 
greatest exposure to mycotoxins is in the handling of infected ears 
and the grinding of infected grain since inhalation is more 
hazardous than ingestion of Fusarium mycotoxins. Gloves, 
coveralls and a dust mask should be worn when harvesting 
because dust and fungal particles are released from the ear as the 
husk is pulled off. Whenever possible, stand upwind when 
handling mold-contaminated ears. When combining 
contaminated field plots, the combine operator should also wear 
protective clothing and a mask, especially if the combine does not 
have a positive-pressure closed-in cab, in which air filters are 
changed frequently. Grinding of contaminated kernels indoors 
should take place in a room equipped with a ventilation /exhaust 
system capable of handling dust removal. Protective clothing 
should be removed and cleaned after use. Hands, face and other 
exposed areas of the body should be washed with soap and water 
before eating. Never eat or drink in any areas where 
contaminated grain or cultures are present. For further 
information, consult the Agriculture and Agri-Food Canada 
publication Reducing Mycotoxins in Animal Feeds (#1827 E). 



36 



References 

Abbas, H.K.; Mirocha, C.J.; Kommedahl, T.; Burnes, P.M.; 
Meronuck, R.A.; and Gunther, R. 1988. Toxigenicity of 
Fusarium proliferation and other Fusariurn species isolated from 
corn ears in Minnesota. Phytopathology 78:1258-1260. 

Atlin, G.N.; Enerson, P.M.; McGirr, L.G.; and Hunter, R.B. 1983. 
Gibberella ear rot development and zearalenone and 
vomitoxin production as affected by maize genotype and 
Gibberella zeae strain. Can. J. Plant Sci. 63:847-853. 

Attwater, W.A.; and Busch, L.V. 1983. Role of the sap beetle 
Glischrochilus quadrisignatus in the epidemiology of gibberella 
corn ear rot. Can. J. Plant Pathol. 5:158-163. 

Charmley, L.L.; and Prelusky, D.B. 1994. Decontamination of 
Fusarium mycotoxins. In: Mycotoxins in Grain: Compounds 
Other Than Aflatoxin. J.D. Miller and H.L. Trenholm (Eds.). 
Eagan Press, St-Paul, Minn. pp. 421-435. 

Chungu, C; Mather, D.E.; Reid, L.M.; and Hamilton, R.I. 1996. 
Comparison of techniques for inoculating maize silk, kernel 
and cob tissues with Fusarium graminearum. Plant Dis. 
80:81-84. 

Cullen, D.; Caldwell, R.W.; and Smalley, E.B. 1983. Susceptibility 
of maize to Gibberella zeae ear rot: relationship to host 
genotype, pathogen virulence, and zearalenone 
contamination. Plant Dis. 67:89-91. 

Enerson, P.M.; and Hunter, R.B. 1980. A technique for screening 
maize {Zea mays L.) for resistance to ear mold incited by 
Gibberella zeae (Schw.) Petch. Can. J. Plant Sci. 60:1123-1128. 

Gordon, W.L. 1959. The occurrence of Fusarium species in 
Canada. VI. Taxonomy and geographic distribution of 
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Hart, L.P; Braselton, J.R.; and Stebbins, T.C. 1982. Production of 
zearalenone and deoxynivalenol in commercial sweet corn. 
Plant Dis. 66:1113-1135. 

Hart, L.P; Casale, W.L.; Gendolf, E.; Rossman, E.; and Isleib, T. 
1987. Resistance of corn inbred lines to Fusarium and Fusarium 
toxins. 42nd Annual Corn and Sorghum Research Conference, 
pp. 161-171. 



37 



Hesseltine, C.W.; and Bothast, R.J. 1977. Mold development in 
ears of corn from tasseling to harvest. Mycologia 69:328-340. 

Koehler, B. 1942. Natural mode of entrance of fungi into corn 
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